Huntington's disease (HD) is a dominant autosomal neurodegenerative disorder caused by an expanded poly-glutamine (polyQ) tract within the amino-terminus of the huntingtin (Htt) protein, whose cellular function remains controversial. To gain insight into Htt function and uncover potential HD pathogenic mechanisms, epitope-tagged Htt was purified from HeLa cells and Argonaute (Ago) proteins were identified as Htt-interacting proteins. Agos have been shown in part to localize to discrete cytoplasmic foci known as P (processing)-bodies. Proteins involved in small RNA-mediated gene silencing, translational repression, mRNA surveillance, and mRNA degradation accumulate with their bound mRNAs at P-bodies, where their destiny is determined. Co-localization studies demonstrated Htt to be present in P-bodies. Furthermore, depletion of Htt showed compromised RNAi-mediated gene silencing, indicating that normal Htt contributes to post-transcriptional regulation of gene expression. Analysis of mouse striatal cells expressing endogenous mutant Htt showed fewer P-bodies and exhibited reduced gene silencing activity compared with their wild-type counterparts. These data suggest that the previously reported transcriptional deregulation in HD may be attributed in part to mutant Htt's role in post-transcriptional processes. The association of Htt with Ago2 and the localization of Htt to P-bodies provide new and compelling evidence connecting Htt with the post-transcriptional control of mRNA abundance and P-body integrity. It is hypothesized that Htt functions as an Ago accessory factor necessary for the regulation of protein levels through RNAi-mediated mechanisms or translational repression/mRNA decay pathways associated with P-bodies. Neuronal RNA granules have been described to be involved in transport and translational control of neuronal mRNAs. A recent study reported the presence of P-body proteins in neuronal granules in Drosophila. It is tempting to speculate that Htt is involved in neuronal RNA granule function, which may be perturbed by mutant Htt, contributing to specificity for neurons in HD. In this proposal the role of wild-type and mutant Htt in post-transcriptional processes will be investigated with a focus on RNA- mediated gene silencing. microRNA reporter assays and functional tethering assays in mouse striatal cells and Htt knockdown or knockout cells will be performed. The distribution of P-bodies in striatal cells as well as in primary neurons expressing endogenous wt or mutant Htt will be determined by immunofluorescence. The purification data indicates that wild-type Htt preferentially associates with Ago2 while mutant Htt associates with TNRC6B, another component of P-bodies involved in gene silencing. It is hypothesized that the Q-rich domains of mutant Htt and TNRC6B contribute to alterations in P-body function. The Htt-TNRC6B interaction will be examined using the yeast two-hybrid system. mRNAs and miRNAs associated with Htt-Ago2 complex will be purified and identified by cDNA microarrays and TaqMan miRNA assays and their biological function as new targets of Htt will be investigated. These experiments should provide new insights to previously uncharacterized role of Htt in post-transcriptional pathways and help to uncover new pathogenic mechanisms that contribute to HD.